We have previously shown effective delivery of a transgene-encoded IFN-alpha protein at the tumor site by combining the use of lentiviral vectors (LVs), capable of efficient gene transfer in hematopoietic stem/progenitor cells (HS/PCs), and the selective activation of the Tie2 promoter in a subset of tumor-infiltrating monocytes (Tie2-expressing monocytes, TEMs), providing the proof of principle of its antitumor efficacy in mice1 . We now aim to increase the safety and feasibility of this strategy toward the goal of a clinical translation. Being TIE2 also expressed by primitive HSPCs, where sustained IFN-alpha expression might be detrimental, we have developed new LVs with de-targeted transgene expression from HS/PCs via microRNA-mediated post-transcriptional regulation. We are currently investigating the safety of this IFN-alpha delivery platform monitoring the HSC long-term repopulating activity in murine and human hematochimeric NSG mice transplanted with gene modified HS/PCs. Moreover we have validated the efficacy of this approach in vivo. Indeed, detargeting of the transgene expression from HS/PCs did not affect the ability of TEM-mediated IFN-alpha delivery to inhibit both primary and secondary spontaneous mammary tumors. By FACS analysis on peripheral blood of transplanted human hematochimeric NSG mice, we showed specific GFP expression in the myeloid compartment, as expected. Most importantly, human Tie2-IFN-alpha myeloid cells were recruited to tumors orthotopically injected into NSG mice, where they effectively targeted IFN-alpha, as shown by the local upregulation of IFN-inducible genes. Altogether our data support the feasibility of a clinical translation of this strategy, which bear the potential of providing a safe and more effective adjuvant treatment against cancer.